Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome regulates ASC speck formation during S. aureus . Fluorescence microscopy images of MAC-T cells immunoassayed for ASC (red) with or without MCC950 after 4 h of S. aureus treatment.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Fluorescence, Microscopy

Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome activation by S. aureus is essential for the generation of GSDMD-N and the release of IL-1β and IL-18. A Activated caspase-1 and B GSDMD-N or released C IL-1β and D IL-18 of MAC-T cells treated with or without NLRP3 inhibitor MCC950 or caspase-1 inhibitor VX765 for 1.5 h prior to treatment with S. aureus for 4 h. E Levels of IL-1β and F IL-18 released after S. aureus treatment of MAC-T cells for 4 h in the presence or absence of 25, 50, or 75 mM KCl.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages

doi: 10.3389/fimmu.2023.1136669

Figure Lengend Snippet: Both NLRP3- and NLRC4-inflammasome activation contributes to rFlaA:Betv1 induced IL-1β and pro-inflammatory cytokine secretion from THP-1 macrophages. PMA-differentiated wild-type (WT), NLRP3-, NLRC4-, or ASC-deficient THP-1 macrophages were stimulated with either LPS as a positive control, or equimolar amounts of rFlaA + rBet v 1, rFlaA:Betv1, rFlaA *D1 :Betv1, or rFlaA ΔDC0 :Betv1 for 24 h (A) . Supernatants were collected and checked for the secretion of IL-1β, IL-6, IL-12, and TNF-α by ELISA (B) . Data are the mean results of three independent experiments ± SD, and statistical significances are indicated as ns: p-value > 0.05, *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001.

Article Snippet: PMA-differentiated, THP-1 macrophages were pre-incubated with the indicated amounts of either the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) or BMS-345541 (Abcam), the unspecific inflammasome inhibitor VX-765 ( Invivo Gen), which inhibits caspase-1 activity, the specific NLRP3-inflammasome inhibitor MCC950 ( Invivo Gen), or the MAPK inhibitors SP600125 (SAP/JNK MAPK inhibitor, Invivo Gen), SB202190 (p38α/β MAPK inhibitor, Invivo Gen), or U0126 (ERK MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with rFlaA:Betv1 for 24 h. The target molecules of the used inhibitors are summarized in .

Techniques: Activation Assay, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages

doi: 10.3389/fimmu.2023.1136669

Figure Lengend Snippet: NFκB- and SAP/JNK MAP kinase-signaling pathways contribute to inflammasome activation by regulating pro-Caspase-1, pro-IL-1β-, and NLRP3-expression in rFlaA:Betv1-stimulated THP-1 macrophages. PMA-differentiated THP-1 macrophages were pre-treated with the indicated inhibitors (500 nM TPCA-1, 5 µM BMS-345541, 25 µM SP600125, or 10 µM SB202190) for 90 minutes, followed by stimulation with 27.4 µg/mL rFlaA:Betv1 for additional 24 h (A) . Proteins in supernatant (Sup) and cell lysate (Lysate) were examined by Western Blot (B) . The intensity of Western Blot bands from three independent experiments was analyzed, first normalized to the loading control β-Tubulin and then again normalized to the unstimulated group (shown as value of “1”) (C) . Data are either representative (B) or mean results of three independent experiments ± SD (C) . Statistical significances are indicated as ns: p-value > 0.05, *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001.

Article Snippet: PMA-differentiated, THP-1 macrophages were pre-incubated with the indicated amounts of either the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) or BMS-345541 (Abcam), the unspecific inflammasome inhibitor VX-765 ( Invivo Gen), which inhibits caspase-1 activity, the specific NLRP3-inflammasome inhibitor MCC950 ( Invivo Gen), or the MAPK inhibitors SP600125 (SAP/JNK MAPK inhibitor, Invivo Gen), SB202190 (p38α/β MAPK inhibitor, Invivo Gen), or U0126 (ERK MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with rFlaA:Betv1 for 24 h. The target molecules of the used inhibitors are summarized in .

Techniques: Activation Assay, Expressing, Western Blot

Journal: Cell communication and signaling : CCS

Article Title: P2X7 receptor activation leads to NLRP3-independent IL-1β release by human macrophages.

doi: 10.1186/s12964-023-01356-1

Figure Lengend Snippet: Fig. 2 P2X7-mediated IL-1β release is NLRP3 independent. A THP-1 macrophages were primed with Pam3CSK4 and then stimulated with ATP, BzATP or nigericin for 3 h. Antagonists of P2X7 receptor (A-804598, oxATP), P2X4 receptor (5-BDBD) or P2X receptors (PPADS) were added 1 h before stimulation. IL-1β concentration in the supernatants was analyzed by ELISA. ATP, BzATP or nigericin induced IL-1β release was set to 100%. Mean + SEM (n = 3). One-sample t test against 100%, ns ≥ 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. B THP-1 macrophages were primed with Pam3CSK4. Potassium chloride was added together with ATP, BzATP or nigericin for 3 h. IL-1β concentration in the supernatants was analyzed by ELISA. ATP, BzATP or nigericin induced IL-1β release was set to 100%. Mean + SEM (n = 3). One-sample t test against 100%, *P ≤ 0.05, ****P ≤ 0.0001. C THP-1 macrophages were primed with Pam3CSK4 and then stimulated with ATP, BzATP or nigericin for 3 h. NLRP3 inhibitors MCC950 or Bay 11–7082 were added 1 h before stimulation. IL-1β concentration in the supernatants was analyzed by ELISA. ATP, BzATP or nigericin induced IL-1β release was set to 100%. Mean + SEM (n = 3—4). One-sample t test against 100%, ns ≥ 0.05, *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001 (D) NLRP3 KO THP-1 macrophages were primed with Pam3CSK4. Cells were then stimulated with ATP, BzATP or nigericin for 3 h. IL-1β concentration in the supernatants was analyzed by ELISA. Mean + SEM (n = 3). Two-tailed two sample t test, ns ≥ 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001

Article Snippet: In selected experiments, cells were preincubated with the non-competitive P2X7 receptor antagonist A804598 (1 μM, 4473, Tocris Bioscience, Bristol United Kingdom), irreversible P2X7 receptor antagonist oxidized ATP (oxATP, 300 μM, 505758, Merck, Darmstadt, Germany), P2X4 receptor antagonist 5-BDBD (25 μM, SML0450-5MG, SigmaAldrich, Taufkirchen, Germany), P2X receptor antagonist PPADS (100 μM, 0625, Tocris Bioscience, Bristol, United Kingdom), NLRP3 inhibitor MCC950 (10 μM, 5479, Tocris Bioscience, Bristol, United Kingdom) or Bay 11–7082 (20 μM, B5556, Sigma-Aldrich, Taufkirchen, Germany), Caspase-1 inhibitor Ac-YVAD-cmk (40 μM, 10014, Biomol, Hamburg, Germany), pan-caspase inhibitor Z-VADfmk (40 μM, tlrl-vad, Invivogen, Toulouse, France), serine protease inhibitor AEBSF (300 μM, 50985.100, Biomol, Hamburg, Germany), cysteine protease inhibitor E64 (10 μM, 324890, Merck, Darmstadt, Germany), aspartic protease inhibitor pepstatin A (50 μM, 2936, Carl Roth, Karlsruhe, Germany) 1 h before stimulation.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cell communication and signaling : CCS

Article Title: P2X7 receptor activation leads to NLRP3-independent IL-1β release by human macrophages.

doi: 10.1186/s12964-023-01356-1

Figure Lengend Snippet: Fig. 4 Influence of priming with different TLR ligands on IL-1β release. A THP-1 macrophages were primed with LPS or Pam2CSK4. Cells were then stimulated with ATP, BzATP or nigericin for 3 h. IL-1β concentration in the supernatants was analyzed by ELISA. Mean + SEM (n = 3). Two-tailed two-sample t test, ns ≥ 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. B THP-1 macrophages were primed with Pam3CSK4, Pam2CSK4 or LPS. Gene expression of IL18 or IL1B was normalized to GAPDH. Mean + SEM (n = 3). One-way ANOVA followed by Tukey´s post-test, ns ≥ 0.05. C THP-1 macrophages were primed with Pam3CSK4, Pam2CSK4 or LPS and then stimulated with ATP, BzATP or nigericin. NLRP3 inhibitor (MCC950) was added 1 h before stimulation. IL-1β concentration in the supernatants was analyzed by ELISA. ATP, BzATP or nigericin induced IL-1β release was set to 100%. Mean + SEM (n = 3—5). One-way ANOVA followed by Tukey´s post-test, ns ≥ 0.05, *P ≤ 0.05, **P ≤ 0.01

Article Snippet: In selected experiments, cells were preincubated with the non-competitive P2X7 receptor antagonist A804598 (1 μM, 4473, Tocris Bioscience, Bristol United Kingdom), irreversible P2X7 receptor antagonist oxidized ATP (oxATP, 300 μM, 505758, Merck, Darmstadt, Germany), P2X4 receptor antagonist 5-BDBD (25 μM, SML0450-5MG, SigmaAldrich, Taufkirchen, Germany), P2X receptor antagonist PPADS (100 μM, 0625, Tocris Bioscience, Bristol, United Kingdom), NLRP3 inhibitor MCC950 (10 μM, 5479, Tocris Bioscience, Bristol, United Kingdom) or Bay 11–7082 (20 μM, B5556, Sigma-Aldrich, Taufkirchen, Germany), Caspase-1 inhibitor Ac-YVAD-cmk (40 μM, 10014, Biomol, Hamburg, Germany), pan-caspase inhibitor Z-VADfmk (40 μM, tlrl-vad, Invivogen, Toulouse, France), serine protease inhibitor AEBSF (300 μM, 50985.100, Biomol, Hamburg, Germany), cysteine protease inhibitor E64 (10 μM, 324890, Merck, Darmstadt, Germany), aspartic protease inhibitor pepstatin A (50 μM, 2936, Carl Roth, Karlsruhe, Germany) 1 h before stimulation.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Gene Expression

Journal: Cell communication and signaling : CCS

Article Title: P2X7 receptor activation leads to NLRP3-independent IL-1β release by human macrophages.

doi: 10.1186/s12964-023-01356-1

Figure Lengend Snippet: Fig. 5 Proposed mechanism for NLRP3-independent IL-1β release by human macrophages after P2X7 receptor activation. Activation of TLR2/1 by Pam3CSK4 leads to NF-κB mediated production of pro-IL-1β and components of NLRP3 inflammasome. Nigericin-mediated K+ efflux leads to NLRP3 inflammasome oligomerization causing serine protease- and caspase-dependent IL-1β release. In contrast, P2X7 receptor stimulation by BzATP potentially initiates two distinct IL-1β releasing mechanisms that are both NLRP3-independent. One mechanism relies on K+ efflux and requires activation of caspase-1 and serine proteases. The other mechanism is independent of K+ efflux, caspases, and serine-, cysteine-, and aspartic proteases, suggesting the involvement of other mechanisms for proteolytic processing of IL-1β

Article Snippet: In selected experiments, cells were preincubated with the non-competitive P2X7 receptor antagonist A804598 (1 μM, 4473, Tocris Bioscience, Bristol United Kingdom), irreversible P2X7 receptor antagonist oxidized ATP (oxATP, 300 μM, 505758, Merck, Darmstadt, Germany), P2X4 receptor antagonist 5-BDBD (25 μM, SML0450-5MG, SigmaAldrich, Taufkirchen, Germany), P2X receptor antagonist PPADS (100 μM, 0625, Tocris Bioscience, Bristol, United Kingdom), NLRP3 inhibitor MCC950 (10 μM, 5479, Tocris Bioscience, Bristol, United Kingdom) or Bay 11–7082 (20 μM, B5556, Sigma-Aldrich, Taufkirchen, Germany), Caspase-1 inhibitor Ac-YVAD-cmk (40 μM, 10014, Biomol, Hamburg, Germany), pan-caspase inhibitor Z-VADfmk (40 μM, tlrl-vad, Invivogen, Toulouse, France), serine protease inhibitor AEBSF (300 μM, 50985.100, Biomol, Hamburg, Germany), cysteine protease inhibitor E64 (10 μM, 324890, Merck, Darmstadt, Germany), aspartic protease inhibitor pepstatin A (50 μM, 2936, Carl Roth, Karlsruhe, Germany) 1 h before stimulation.

Techniques: Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Andrade-Oliveira Salvianolic Acid B Modulates Caspase-1–Mediated Pyroptosis in Renal Ischemia-Reperfusion Injury via Nrf2 Pathway

doi: 10.3389/fphar.2020.541426

Figure Lengend Snippet: Salvianolic acid B (SalB) promotes Nrf2 nuclear activation and inhibits NLR family pyrin domain-containing 3 (NLRP3)/thioredoxin-interacting protein-thioredoxin1 (TXNIP) expression in ischemia-reperfusion (I/R) mice. (A–C) The expression of NLRP3 and TXNIP. (D) Immunofluorescence images (magnification ×200) showing the nuclear expression and localization of Nrf2 in the Sham, I/R, SalB-L, SalB-M, SalB-H groups. Blue: nuclear staining (DAPI); red: Nrf2; staining. Scale bar: 20 μm. (E) Representative western blots and (F–H) quantification of relative protein expression for nuclear Nrf2, keap1 and HO-1. (I–K) Superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) detected by a microplate reader. Data are represented as images or expressed as the mean ± SEM of each group from three separate experiments. *p < 0.05, **p < 0.01 vs. sham group; # p < 0.05, ## p < 0.01 vs. I/R group.

Article Snippet: After two–four passages, cells were cultured for 24 h and then were randomly divided into three groups: Control group: cells were cultured under normal conditions (5% CO2, 21% O2, 74% N2); Model group: the cells were incubated with different concentrations of SalB(purity>98%; 358153; Nanjing DASF Biotechnology Co.Ltd.) for 24 h; Positive control group: cells were incubated in certain concentration of NLRP3 inhibitor MCC950(S7809;Selleck), caspase-1 specific inhibitor VX-765(S2228;Selleck) for 2 h, and were then placed in a hypoxia incubator chamber (27310, stemcell) hypoxia (5% CO2, 95% N2) for 6 h and then reoxygenated for an additional 1 h (5% CO2, 21% O2, 74% N2).

Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Andrade-Oliveira Salvianolic Acid B Modulates Caspase-1–Mediated Pyroptosis in Renal Ischemia-Reperfusion Injury via Nrf2 Pathway

doi: 10.3389/fphar.2020.541426

Figure Lengend Snippet: Nuclear factor erythroid-2 related factor 2 (Nrf2) nuclear expression is upregulated and NLR family pyrin domain-containing 3 (NLRP3)/thioredoxin-interacting protein-thioredoxin1 (TXNIP) is down-regulated after SalB treatment in H/R (A–C) NLRP3 and TXNIP expression were examined by western blot. (D) Immunofluorescence results (magnification ×400) showing the expression of Nrf2 under normal conditions (control), SalB treatment and H/R-treated HK-2 cells. Blue, nuclear staining (DAPI); red, Nrf2 staining. Scale bar: 20 μm. (E) Representative western blots and (F–H) quantification of relative protein expression for nuclear Nrf2, keap1 and HO-1. (I) Representative images of fluorescence of ROS probed by DCFH-DA. Data are represented as images or expressed as the mean ± SEM of each group from three separate experiments. **p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. H/R group.

Article Snippet: After two–four passages, cells were cultured for 24 h and then were randomly divided into three groups: Control group: cells were cultured under normal conditions (5% CO2, 21% O2, 74% N2); Model group: the cells were incubated with different concentrations of SalB(purity>98%; 358153; Nanjing DASF Biotechnology Co.Ltd.) for 24 h; Positive control group: cells were incubated in certain concentration of NLRP3 inhibitor MCC950(S7809;Selleck), caspase-1 specific inhibitor VX-765(S2228;Selleck) for 2 h, and were then placed in a hypoxia incubator chamber (27310, stemcell) hypoxia (5% CO2, 95% N2) for 6 h and then reoxygenated for an additional 1 h (5% CO2, 21% O2, 74% N2).

Techniques: Expressing, Western Blot, Immunofluorescence, Control, Staining, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Andrade-Oliveira Salvianolic Acid B Modulates Caspase-1–Mediated Pyroptosis in Renal Ischemia-Reperfusion Injury via Nrf2 Pathway

doi: 10.3389/fphar.2020.541426

Figure Lengend Snippet: The pyroptosis of acute kidney injury (AKI) signaling and protective effect of SalB through the nuclear factor erythroid-2 related factor 2 (Nrf2)/NLR family pyrin domain-containing 3 (NLRP3) pathway. I/R and H/R trigger pyroptotic cell death signaling, including upregulation of thioredoxin-interacting protein-thioredoxin1 (TXNIP) and suppression of Nrf2 expression, leads to NLRP3 oligomerization, ASC recruitment and subsequent caspase-1 activation. Fortunately, SalB treatment ameliorates AKI by regulating the Nrf2/NLRP3 pathway. The arrows represent promotion, while the inverted T represent inhibition. The effect of Salvianolic acid B (SalB) is shown in red.

Article Snippet: After two–four passages, cells were cultured for 24 h and then were randomly divided into three groups: Control group: cells were cultured under normal conditions (5% CO2, 21% O2, 74% N2); Model group: the cells were incubated with different concentrations of SalB(purity>98%; 358153; Nanjing DASF Biotechnology Co.Ltd.) for 24 h; Positive control group: cells were incubated in certain concentration of NLRP3 inhibitor MCC950(S7809;Selleck), caspase-1 specific inhibitor VX-765(S2228;Selleck) for 2 h, and were then placed in a hypoxia incubator chamber (27310, stemcell) hypoxia (5% CO2, 95% N2) for 6 h and then reoxygenated for an additional 1 h (5% CO2, 21% O2, 74% N2).

Techniques: Expressing, Activation Assay, Inhibition

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of AZD4144, a Selective and Potent NLRP3 Inhibitor for the Treatment of Inflammatory Diseases

doi: 10.1021/acs.jmedchem.5c00781

Figure Lengend Snippet: MCC950/CP-456773.

Article Snippet: The NLRP3 inflammasome is a central driver of downstream generation of pro-inflammatory mediators such as IL-1β and IL-18 , For the development of targeted therapies, there is no question that NLRP3 stands out as a key target to modulate − and selective inhibition of NLRP3 is hypothesized to be a promising treatment option for a large number of diseases connected to inflated inflammatory response., − The archetypal NLRP3 inhibitor MCC950, also known as CP-456773 ( ), was originally discovered by Pfizer in the late 1990s.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of AZD4144, a Selective and Potent NLRP3 Inhibitor for the Treatment of Inflammatory Diseases

doi: 10.1021/acs.jmedchem.5c00781

Figure Lengend Snippet: Concentration response curves from cell-based NanoBRET tracer competition binding experiments. Competition of MCC950, 4 , 17 , 21 , and 25 against (A) an MCC950 analogue-based tracer or (B) tracer 28 based on 4 . Potency values and numbers of separate experiments are shown in . Error bars represent SD for responses at each concentration. Colors and labels are as indicated in the figure.

Article Snippet: The NLRP3 inflammasome is a central driver of downstream generation of pro-inflammatory mediators such as IL-1β and IL-18 , For the development of targeted therapies, there is no question that NLRP3 stands out as a key target to modulate − and selective inhibition of NLRP3 is hypothesized to be a promising treatment option for a large number of diseases connected to inflated inflammatory response., − The archetypal NLRP3 inhibitor MCC950, also known as CP-456773 ( ), was originally discovered by Pfizer in the late 1990s.

Techniques: Concentration Assay, Binding Assay

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of AZD4144, a Selective and Potent NLRP3 Inhibitor for the Treatment of Inflammatory Diseases

doi: 10.1021/acs.jmedchem.5c00781

Figure Lengend Snippet: Concentration–response curves of MCC950, 4 , 17 , 21 , and 25 in (A) nigericin-triggered speck formation, (B) nigericin-triggered IL-1β, (C) ATP-triggered IL-1β, and (D) poly­(dA:dT)-triggered IL-1β production (AIM2 selectivity). Colors and symbols are as indicated in the figure. Potency values and numbers of separate experiments are shown in . Error bars represent SD for responses at each concentration.

Article Snippet: The NLRP3 inflammasome is a central driver of downstream generation of pro-inflammatory mediators such as IL-1β and IL-18 , For the development of targeted therapies, there is no question that NLRP3 stands out as a key target to modulate − and selective inhibition of NLRP3 is hypothesized to be a promising treatment option for a large number of diseases connected to inflated inflammatory response., − The archetypal NLRP3 inhibitor MCC950, also known as CP-456773 ( ), was originally discovered by Pfizer in the late 1990s.

Techniques: Concentration Assay

Journal: Journal of Neuroinflammation

Article Title: Fenebrutinib, a Bruton’s tyrosine kinase inhibitor, blocks distinct human microglial signaling pathways

doi: 10.1186/s12974-024-03267-5

Figure Lengend Snippet: Human microglial pathways not impacted by fenebrutinib treatment. A Cytokine and chemokine release from LPS-stimulated (0.1 μg/mL) human iMicroglia incubated with fenebrutinib or ibrutinib (both 1 μM) for 24 h. While BTK inhibition reduced levels of GM-CSF and CCL17, limited effects were observed for other cytokines and chemokines that are increased with LPS stimulation. B IL-1β release from human iMicroglia incubated with fenebrutinib or MCC-950 (0.01–1 μM) and stimulated with LPS (0.1 μg/mL; 3 h) followed by nigericin (10 μM; 1 h). Fenebrutinib had no effect on IL-1β release in contrast to MCC-950, which dose dependently reduced IL-1β levels. C Phagocytosis of pHrodo-conjugated myelin by human iMicroglia incubated with fenebrutinib or ibrutinib (0.01–1 μM) for 24 h. Fenebrutinib had no effect on myelin phagocytosis, while ibrutinib reduced myelin phagocytosis at 1 μM only. Data are shown as mean ± SD; individual dots represent replicate wells. Results are representative of two to three independent experiments. Significance is indicated by **** P < 0.0001, *** P < 0.001 and * P < 0.05, determined by one‐way ANOVA and Tukey’s post hoc test. ANOVA analysis of variance, BTK Bruton’s tyrosine kinase, CCL C-C motif chemokine ligand, CXCL C-X-C motif chemokine ligand, GM-CSF granulocyte–macrophage colony-stimulating factor, FEN fenebrutinib, IL interleukin, iMicroglia induced pluripotent stem cell-derived microglia, LPS lipopolysaccharide, NLRP3 NACHT, LRR and PYD domains-containing protein 3, SD standard deviation, TNF tumor necrosis factor

Article Snippet: Cells were stimulated with lipopolysaccharide (LPS from E. coli, Serotype O55:B5, Enzo Life Sciences, ALX-581-013; 1 μg/mL) for 3 h. NLRP3 inhibitor tool compound MCC-950 (CP-456773 sodium salt; Selleckchem, S7809; 0.01–1 μM), fenebrutinib (0.01–1 μM) or vehicle (DMSO; Sigma, D2650) were added prior to nigericin (10 μM; Invivogen, tlrl-nig-5) addition.

Techniques: Incubation, Inhibition, Derivative Assay, Standard Deviation